Project Summary Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for many patients with hematologic disease or leukemia. In 2015, >22,000 HSCT were performed in the United States. Donor HSC experience many stresses during transplant, including myelo-toxic conditioning that alters the niche, ex vivo manipulation, and supra-physiological expansion. Although post-transplant stress hematopoiesis is likely subject to biological regulation distinct from steady-state hematopoiesis, the molecular regulation of HSCT is poorly understood. We seek to identify the key intrinsic regulators of HSCT. In a screen for genes whose depletion perturbs murine HSPC in vivo repopulation, we identified GASP (G-protein coupled receptor (GPCR) Associated Sorting Protein) family member, Gprasp2, as a novel negative regulator of HSCT. We discovered that loss of Gprasp2, or the highly related gene, Gprasp1, dramatically enhances HSPC competitive repopulating activity. Gprasp1 and Gprasp2 are both expressed by murine and human HSC. GPRASP1 and GPRASP2 bind a motif in the C-terminus of GPCRs to traffic them to lysosomes. Thus, loss of GPRASP1 and GPRASP2 likely stabilizes GPCRs that promote HSCT. CXCR4, a master regulator of HSC migration, survival and quiescence, contains a GASP-binding motif and is thus a putative GASP target. Indeed, Gprasp1 or Gprasp2 knockdown reduced apoptosis, increased quiescence and stabilized CXCR4 in HSPC ex vivo and acutely post-transplant. These phenotypes were abolished in Cxcr4-/- HSPC. Thus, these data implicate GASP family members as novel regulators of CXCR4. We hypothesize that GPRASP1 and GPRASP2 loss boosts HSCT by promoting HSPC survival and quiescence via CXCR4 stabilization. We will test this and also assess a role for GASPs in native hematopoiesis, according to the following Aims: 1) to illuminate cellular mechanisms that enhance HSC transplantation, 2) to identify molecular targets whose stabilization enhances HSC transplantation and 3) to identify novel molecular bottlenecks of human HSC transplantation. In Aim 1, we will use Gprasp1 and Gprasp2 shRNAs to examine Gprasp-deficient HSPC for apoptosis, quiescence, and niche retention acutely post- transplant. We will also use SclERT2-CreGprasp1fl/fl and SclERT2-CreGprasp2fl/fl mice to interrogate a role for Gprasp1 and Gprasp2 in steady-state hematopoiesis. In Aim 2, we will employ Cxcr4fl/flROSA26Cre-ERT2 mice to test if CXCR4 is a key functional target of GPRASP1 or GPRASP2 in HSPC. We will also examine CXCR4 stability and localization in Gprasp-deficient HSPCs and use standard biochemical assays to test for a physical interaction between GPRASP1, GPRASP2 and CXCR4. Finally, in Aim 3, we will treat human CD34+ HSPC with GPRASP1 or GPRASP2-shRNAs to test if GPRASP loss can enhance bone marrow engraftment of human cells when transplanted into NOD-scid IL2r?null mice. This work will cement GASP family members as novel negative regulators of HSPC, reveal a new mechanism of CXCR4 regulation in HSC, and illuminate new molecular targets for improving HSCT.